灭字脊虎天牛触角转录组中气味结合蛋白基因的鉴定及表达谱分析
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摘要
【目的】发掘灭字脊虎天牛(Xylotrechus quadripes Chevrolat)成虫触角中的OBP基因,明确OBP基因在成虫不同组织的表达情况。【方法】采用Illumina HiSeq~(TM) 2000测序平台对灭字脊虎天牛的触角进行转录组测序,利用BLAST同源搜索的方法鉴定OBP基因,采用RT-PCR技术研究OBP基因的表达谱。【结果】从灭字脊虎天牛的触角转录组中共鉴定到24个OBP基因,所有OBP基因均具有全长开放阅读框(123~180个氨基酸)和信号肽序列(16~23个氨基酸)。多序列比对结果表明,不同OBP间一致性较低,其平均值为19.4%。序列比对和进化树分析结果表明,9个OBP具有6个保守的半胱氨酸,属于Classic OBPs家族;15个OBP具有4个保守的半胱氨酸,属于Minus-C OBPs家族。RT-PCR结果表明,Xqua OBP4、OBP8、OBP18和OBP21在触角特异表达;Xqua OBP2、OBP3、OBP7和OBP20在触角高表达,另外在其他组织中也有表达;其他OBPs基因在检测的两个或多个组织中均有不同程度的表达。【结论】从灭字脊虎天牛触角转录组中共鉴定得到24个OBP基因,其中部分基因在触角特异或高表达,很可能参与嗅觉功能;部分基因在非嗅觉组织中有表达,很可能具有非嗅觉功能。
【Objective】The objective of the study was to identity odorant binding protein(OBP) genes in adult antennae of Xylotrechus quadripes Chevrolat and to explore their expression patterns in different tissues.【Method】The transcriptome of X. quadripes was sequenced using Illumina HiSeq~(TM) 2000. Further,a homology-based method was employed to identify OBP genes from the transcriptome. The expression patterns of OBPs were determined by reverse transcription PCR(RT-PCR). 【Result】 24 transcripts encoding putative OBPs were identified from X. quadripes antennal transcriptome, all of which shared full-length open reading frames(120~180 amino acids) and signal peptides at N-terminal(16~23 amino acids). Multiple sequence alignment results showed that a low identity was observed among different OBPs,with an average protein identity of 19.4%. Analyses of sequence alignment and phylogenetic tree showed that 9 OBPs had six conserved cysteine(Cys) residues,and belonged to Classic OBPs family;15 OBPs had four conserved Cys residues and belonged to Minus-C OBPs family. The results of RT-PCR showed that Xqua OBP4,OBP8,OBP18 and OBP21 were specifically expressed in antennae;Xqua OBP2,OBP3,OBP7 and OBP20 were highly expressed in the antennae,and slightly in other tissues;other OBPs were expressed in at least two tissue types.【Conclusion】Based on the antennal transcriptome of X.quadripes,24 OBP genes were identified. Expression pattern analysis revealed that some OBP genes were specifically or highly expressed in antennae,possibly having olfactory functions;whereas some were expressed in non-olfactory tissues,possibly guiding non-olfactory behaviors.
【Objective】The objective of the study was to identity odorant binding protein(OBP) genes in adult antennae of Xylotrechus quadripes Chevrolat and to explore their expression patterns in different tissues.【Method】The transcriptome of X. quadripes was sequenced using Illumina HiSeq~(TM) 2000. Further,a homology-based method was employed to identify OBP genes from the transcriptome. The expression patterns of OBPs were determined by reverse transcription PCR(RT-PCR). 【Result】 24 transcripts encoding putative OBPs were identified from X. quadripes antennal transcriptome, all of which shared full-length open reading frames(120~180 amino acids) and signal peptides at N-terminal(16~23 amino acids). Multiple sequence alignment results showed that a low identity was observed among different OBPs,with an average protein identity of 19.4%. Analyses of sequence alignment and phylogenetic tree showed that 9 OBPs had six conserved cysteine(Cys) residues,and belonged to Classic OBPs family;15 OBPs had four conserved Cys residues and belonged to Minus-C OBPs family. The results of RT-PCR showed that Xqua OBP4,OBP8,OBP18 and OBP21 were specifically expressed in antennae;Xqua OBP2,OBP3,OBP7 and OBP20 were highly expressed in the antennae,and slightly in other tissues;other OBPs were expressed in at least two tissue types.【Conclusion】Based on the antennal transcriptome of X.quadripes,24 OBP genes were identified. Expression pattern analysis revealed that some OBP genes were specifically or highly expressed in antennae,possibly having olfactory functions;whereas some were expressed in non-olfactory tissues,possibly guiding non-olfactory behaviors.
引文
[1]BRUYNE M D,BAKER T C.Odor Detection in Insects:Volatile Codes[J].Journal of Chemical Ecology,2008,34(7):882-897.
[2]SCHNEIDER D.Insect antennae[J].Annual Review of Entomology,1964,9(1):103-122.
[3]SATO K,TOUHARA K.Insect olfaction:receptors,signal transduction and behavio[rJ].Results&Problems in Cell Differentiation,2009(47):121-138.
[4]PELOSI P,CALVELLO M,BAN L.Diversity of odorant-binding proteins and chemosensory proteins in insects[J].Chemical Senses,2005,30(1):291-292.
[5]XU Y L,HE P,ZHANG L,et al.Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects[J].BMC Genomics,2009,10(1):632.
[6]LEAL W S,NIKONOVA L,PENG G.Disulfide structure of the pheromone binding protein from the silkworm moth,Bombyx mor[iJ].Febs Letters,1999,464(1-2):85-90.
[7]PELOSI P,ZHOU J J,BAN L P,et al.Soluble proteins in insect chemical communication[J].Cellular&Molecular Life Science,2006,63(14):1658-1676.
[8]ZHOU J J.Odorant-binding proteins in insects[J].Vitamins&Hormones,2010(83):241-272.
[9]VOGT R G,RIDDIFORD L M.Pheromone binding and inactivation by moth antenna[J].Nature,1981,293(5828):161-163.
[10]ZHOU J J,ROBERTSON G,HE X L,et al.Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components[J].Journal of Molecular Biology,2009,389(3):529-545.
[11]ZHANG G H,LI Y P,XU X L,et al.Identification and characterization of two general odorant binding protein genes from the oriental fruit moth,Grapholita molesta,(Busck)[J].Journal of Chemical Ecology,2012,38(4):427-436.
[12]PEARCE S L,CLARKE D F,EAST P D,et al.Genomic innovations,transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa,pest species[J].BMC Biology,2017,15(1):63.
[13]HEKMATSCAFE D S,SCAFE C R,MCKINNEY A J,et al.Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster[J].Genome Research,2002,12(9):1357-1369.
[14]WEI Y,LI L,QIN W,et al.Identification and tissue expression profiling of odorant binding protein genes in the red palm weevil,Rhynchophorus ferrugineus[J].Springerplus,2016,5(1):1542.
[15]GU X C,ZHANG Y N,KANG K,et al.Antennal transcriptome analysis of odorant reception genes in the red turpentine beetle(RTB),Dendroctonus valens[J].PLo S One,2015,10(5):e0125159.
[16]RICHARDS S,GIBBS R A,WEINSTOCK G M,et al.The genome of the model beetle and pest Tribolium castaneum[J].Nature,2008,452(7190):949-955.
[17]GRAHAM L A,TANG W,BAUST J G,et al.Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins[J].Insect Biochemistry&Molecular Biology,2001,31(6-7):691-702.
[18]GAO X,WANG M Q.A c DNA library from the antenna of Monochamus alternatus Hope and binding properties of odorantbinding proteins[J].Journal of Applied Entomology,2015,139(3):229-236.
[19]HU P,WANG J,CUI M,et al.Antennal transcriptome analysis of the Asian longhorned beetle Anoplophora glabripennis[J].Scientific Reports,2016(6):26652.
[20]李广伟,陈秀琳,尚天翠.黄斑星天牛雌虫触角气味结合蛋白基因的鉴定及组织分布[J].生态学杂志,2017,36(6):1678-1689.
[21]FILOMENA,BIASIO,RIVIELLO,et al.Expression pattern analysis of odorant-binding proteins in the pea aphid Acyrthosiphon pisum[J].Insect Science,2015,22(2):220-234.
[22]YUAN H B,DING Y X,GU S H,et al.Molecular characterization and expression profiling of odorant-binding proteins in Apolygus lucorum[J].PLo S One,2015,10(10):e0140562.
[23]XU P,ATKINSON R,JONES D N,et al.Drosophila OBPLUSH is required for activity of pheromone-sensitive neurons[J].Neuron,2005,45(2):193-200.
[24]GHANIM M,DOMBROVSKY A,RACCAH B,et al.A microarray approach identifies ANT,OS-D and takeout-like genes as differentially regulated in alate and apterous morphs of the green peach aphid Myzus persicae(Sulzer[)J].Insect Biochemistry&Molecular Biology,2006,36(11):857-868.
[25]周又生,赵忠喜,李松林,等.咖啡灭字虎天牛生物生态学及发生危害规律和治理研究[J].西南大学学报(自然科学版),2002,24(1):1-4.
[26]舒梅,成文章,李忠恒,等.思茅咖啡灭字脊虎天牛发生与危害调查[J].热带农业科学,2016,36(5):81-84.
[27]舒梅,李忠恒,成文章,等.普洱咖啡天牛的种群动态及优势种群差异研究[J].中国热带农业,2016(2):42-47.
[28]杨文波,吴国星,徐志强,等.管氏肿腿蜂对咖啡灭字脊虎天牛寄生作用的研究[J].环境昆虫学报,2017,39(2):405-410.
[29]周又生,王华,周庆辉,等.咖啡旋皮天牛与咖啡灭字虎天牛发生危害比较研究[J].西南大学学报(自然科学版),2003,25(1):24-27.
[30]ZHU J,ARENA S,SPINELLI S,et al.Reverse chemical ecology:olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles[J].Proc Natl Acad Sci U S A,2017,114(46):e9802-e9810.
[31]PETERSEN T N,BRUNAK S,VON H G,et al.Signal P 4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786.
[32]SAITOU N,NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology&Evolution,1987,4(4):406-25.
[33]王桂荣,郭予元,吴孔明.昆虫触角气味结合蛋白的研究进展[J].昆虫学报,2002,45(1):131-137.
[34]范贤林,卢美光,孟香清,等.棉铃虫室内饲养技术的改进[J].应用昆虫学报,2003,40(1):85-87.
[35]王伟,李秀环,周超,等.甜菜夜蛾人工饲养技术优化[J].环境昆虫学报,2011,33(1):95-98.
[36]魏娟,肖铁光,张友军.小菜蛾室内继代饲养技术及抗性选育[J].作物研究,2009,23(2):129-132.
[37]孙庚,刘少武,常秀辉,等.一种改进的斜纹夜蛾人工饲养技术的效果研究[J].山东农业科学,2015,47(2):104-106.
[38]LI H,ZHANG A,CHEN L Z,et al.Construction and analysis of c DNA libraries from the antennae of Batocera horsfieldi and expression pattern of putative odorant binding proteins[J].Journal of Insect Science,2014,14(57):1-15.
[39]LI X M,ZHU X Y,WANG Z Q,et al.Candidate chemosensory genes identified in Colaphellus bowringiby antennal transcriptome analysis[J].BMC Genomics,2015,16(1):1028.
[40]ANDERSSON M N,GROSSEWILDE E,KEELING C I,et al.Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles,Ips typographus and Dendroctonus ponderosae(Coleoptera:Curculionidae:Scolytinae)[J].BMC Genomics,2013,14(1):198.
[2]SCHNEIDER D.Insect antennae[J].Annual Review of Entomology,1964,9(1):103-122.
[3]SATO K,TOUHARA K.Insect olfaction:receptors,signal transduction and behavio[rJ].Results&Problems in Cell Differentiation,2009(47):121-138.
[4]PELOSI P,CALVELLO M,BAN L.Diversity of odorant-binding proteins and chemosensory proteins in insects[J].Chemical Senses,2005,30(1):291-292.
[5]XU Y L,HE P,ZHANG L,et al.Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects[J].BMC Genomics,2009,10(1):632.
[6]LEAL W S,NIKONOVA L,PENG G.Disulfide structure of the pheromone binding protein from the silkworm moth,Bombyx mor[iJ].Febs Letters,1999,464(1-2):85-90.
[7]PELOSI P,ZHOU J J,BAN L P,et al.Soluble proteins in insect chemical communication[J].Cellular&Molecular Life Science,2006,63(14):1658-1676.
[8]ZHOU J J.Odorant-binding proteins in insects[J].Vitamins&Hormones,2010(83):241-272.
[9]VOGT R G,RIDDIFORD L M.Pheromone binding and inactivation by moth antenna[J].Nature,1981,293(5828):161-163.
[10]ZHOU J J,ROBERTSON G,HE X L,et al.Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components[J].Journal of Molecular Biology,2009,389(3):529-545.
[11]ZHANG G H,LI Y P,XU X L,et al.Identification and characterization of two general odorant binding protein genes from the oriental fruit moth,Grapholita molesta,(Busck)[J].Journal of Chemical Ecology,2012,38(4):427-436.
[12]PEARCE S L,CLARKE D F,EAST P D,et al.Genomic innovations,transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa,pest species[J].BMC Biology,2017,15(1):63.
[13]HEKMATSCAFE D S,SCAFE C R,MCKINNEY A J,et al.Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster[J].Genome Research,2002,12(9):1357-1369.
[14]WEI Y,LI L,QIN W,et al.Identification and tissue expression profiling of odorant binding protein genes in the red palm weevil,Rhynchophorus ferrugineus[J].Springerplus,2016,5(1):1542.
[15]GU X C,ZHANG Y N,KANG K,et al.Antennal transcriptome analysis of odorant reception genes in the red turpentine beetle(RTB),Dendroctonus valens[J].PLo S One,2015,10(5):e0125159.
[16]RICHARDS S,GIBBS R A,WEINSTOCK G M,et al.The genome of the model beetle and pest Tribolium castaneum[J].Nature,2008,452(7190):949-955.
[17]GRAHAM L A,TANG W,BAUST J G,et al.Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins[J].Insect Biochemistry&Molecular Biology,2001,31(6-7):691-702.
[18]GAO X,WANG M Q.A c DNA library from the antenna of Monochamus alternatus Hope and binding properties of odorantbinding proteins[J].Journal of Applied Entomology,2015,139(3):229-236.
[19]HU P,WANG J,CUI M,et al.Antennal transcriptome analysis of the Asian longhorned beetle Anoplophora glabripennis[J].Scientific Reports,2016(6):26652.
[20]李广伟,陈秀琳,尚天翠.黄斑星天牛雌虫触角气味结合蛋白基因的鉴定及组织分布[J].生态学杂志,2017,36(6):1678-1689.
[21]FILOMENA,BIASIO,RIVIELLO,et al.Expression pattern analysis of odorant-binding proteins in the pea aphid Acyrthosiphon pisum[J].Insect Science,2015,22(2):220-234.
[22]YUAN H B,DING Y X,GU S H,et al.Molecular characterization and expression profiling of odorant-binding proteins in Apolygus lucorum[J].PLo S One,2015,10(10):e0140562.
[23]XU P,ATKINSON R,JONES D N,et al.Drosophila OBPLUSH is required for activity of pheromone-sensitive neurons[J].Neuron,2005,45(2):193-200.
[24]GHANIM M,DOMBROVSKY A,RACCAH B,et al.A microarray approach identifies ANT,OS-D and takeout-like genes as differentially regulated in alate and apterous morphs of the green peach aphid Myzus persicae(Sulzer[)J].Insect Biochemistry&Molecular Biology,2006,36(11):857-868.
[25]周又生,赵忠喜,李松林,等.咖啡灭字虎天牛生物生态学及发生危害规律和治理研究[J].西南大学学报(自然科学版),2002,24(1):1-4.
[26]舒梅,成文章,李忠恒,等.思茅咖啡灭字脊虎天牛发生与危害调查[J].热带农业科学,2016,36(5):81-84.
[27]舒梅,李忠恒,成文章,等.普洱咖啡天牛的种群动态及优势种群差异研究[J].中国热带农业,2016(2):42-47.
[28]杨文波,吴国星,徐志强,等.管氏肿腿蜂对咖啡灭字脊虎天牛寄生作用的研究[J].环境昆虫学报,2017,39(2):405-410.
[29]周又生,王华,周庆辉,等.咖啡旋皮天牛与咖啡灭字虎天牛发生危害比较研究[J].西南大学学报(自然科学版),2003,25(1):24-27.
[30]ZHU J,ARENA S,SPINELLI S,et al.Reverse chemical ecology:olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles[J].Proc Natl Acad Sci U S A,2017,114(46):e9802-e9810.
[31]PETERSEN T N,BRUNAK S,VON H G,et al.Signal P 4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786.
[32]SAITOU N,NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology&Evolution,1987,4(4):406-25.
[33]王桂荣,郭予元,吴孔明.昆虫触角气味结合蛋白的研究进展[J].昆虫学报,2002,45(1):131-137.
[34]范贤林,卢美光,孟香清,等.棉铃虫室内饲养技术的改进[J].应用昆虫学报,2003,40(1):85-87.
[35]王伟,李秀环,周超,等.甜菜夜蛾人工饲养技术优化[J].环境昆虫学报,2011,33(1):95-98.
[36]魏娟,肖铁光,张友军.小菜蛾室内继代饲养技术及抗性选育[J].作物研究,2009,23(2):129-132.
[37]孙庚,刘少武,常秀辉,等.一种改进的斜纹夜蛾人工饲养技术的效果研究[J].山东农业科学,2015,47(2):104-106.
[38]LI H,ZHANG A,CHEN L Z,et al.Construction and analysis of c DNA libraries from the antennae of Batocera horsfieldi and expression pattern of putative odorant binding proteins[J].Journal of Insect Science,2014,14(57):1-15.
[39]LI X M,ZHU X Y,WANG Z Q,et al.Candidate chemosensory genes identified in Colaphellus bowringiby antennal transcriptome analysis[J].BMC Genomics,2015,16(1):1028.
[40]ANDERSSON M N,GROSSEWILDE E,KEELING C I,et al.Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles,Ips typographus and Dendroctonus ponderosae(Coleoptera:Curculionidae:Scolytinae)[J].BMC Genomics,2013,14(1):198.